Question: Validation of Splicing Results from cufflinks-cuffdiff pipeline
4
gravatar for pm2012
4.9 years ago by
pm201280
United States
pm201280 wrote:

Hi

I am shortlisting my candidates for validation using qPCR/RTPCR of my results from two different splicing pipelines(DEXSeq & Cufflinks/Cuffdiff). However, I am a little lost as to what candidates I should pick to validate alternative splicing between two conditions/samples. I have picked up candidate exons for validation from DEXSeq results. However, just validating at the exon level seems insufficient to me and I want to validate at the transcript level. I was analyzing potential candidates from my cuffdiff results to find interesting transcripts. However, I am having trouble finding good candidates. 

Here's my approach:

Fist I looked at the results from splicing.diff and identified genes that show statistically significant alternative splicing (based on pvalue of the JS metric). From here I identified the tss_id of the candidate gene (picked one to start if multiple present). Using this tss_id, I went on to identify the transcripts that have this tss_id (in theory spliced from sample primary transcript or promoter). Then, I checked the tss_id in tss_group_exp.diff file to see if it showed differntial expression ( assuming differential promoter usage). If the tss_id was differentially expressed(statistically significantly) then I would assume the difference was because of promoter usage and not splicing per se. So I discarded the candidate. If tss_id wasn't differentially expressed I moved on to check the transcripts belonging to this tss_id in isoform_exp.diff file. Interesting candidates would be those transcripts that showed statistically significant differential splicing at this level. However, I barely found any candidates that matched my criteria. So am in a limbo.

I ran cufflinks using gtf as I was only interested in identifying the shift in isoform ratio of known transcripts due to alternative splicing. I do understand that one possible issue could be because of the presence of novel transcripts not reflected in the results of my pipeline. However I would really appreciate any insights or correction of my logic for finding validation candidates. A great help would be pointing at papers that have done so using similar approach/pipeline. I didn't find any so far.

Thanks 

 

ADD COMMENTlink modified 4.9 years ago • written 4.9 years ago by pm201280
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1875 users visited in the last hour